The epigenetic system of eukaryotic organisms can be defined as the set of chromatin-based processes that establish global gene expression states and promote the long-term maintenance and/or heritability of such states. Most modern work in the field has focused on histone post- translational modifications (PTMs) and the enzymes that impart and remove these modifications as a means of understanding how gene expression states can be imposed and subsequently propagated. However, recent results indicate that the dynamic processes of nucleosome assembly, eviction and replacement are key to epigenetic inheritance. To explore nucleosome dynamics at high resolution, we propose to develop a novel and general technology for the genome-wide measurement of histone replacement kinetics on timescales that have been previously intractable. This technology, which we refer to as Covalent Attachment of Tags to Capture Histones and Identify Turnover (CATCH-IT), will utilize co-translational incorporation of the methionine surrogate azidohomoalanine (AHA) into proteins, followed by isolation of chromatin and in vitro ligation of a biotin moiety to AHA-bearing nucleosomes. This metabolic labeling strategy will be followed by streptavidin affinity pulldown of tagged nucleosomes containing newly synthesized histones, allowing measurement of histone replacement rates across the genome as well as analysis of PTMs on newly synthesized histones. The CATCH-IT method will be developed in the Drosophila S2 cell line but could be applied to any cell type given the generic nature of its component techniques and the fact that no transgenes or antibodies are required. Whereas current ChIP-chip methods offer only a relatively static view of chromatin-based processes, the CATCH-IT method will allow the rates of histone turnover to be measured throughout the genome, adding a temporal dimension to epigenomics research. PUBLIC HEALTH RELEVANCE: We propose to develop a novel technology for measuring the kinetics of chromatin-based processes and to apply it to key epigenetic signatures. This will provide a timeline for dynamic regulation of chromatin using a procedure that is generally applicable to different organisms, cell types and tissues. As such, our technology has the potential of revolutionizing epigenomics research, including efforts that are aimed at diagnosis and treatment of cancer and other epigenetic disorders.